Our laboratory has embarked on a systematic analysis of p15INK4b, which in human acute myeloid leukemia (AML) is inactivated more frequently than any other tumor suppressor gene with cell cycle regulatory properties. p15Ink4b is a cyclin-dependent kinase inhibitor and like other Ink4b family members binds to early G1 cyclin-dependent kinases 4 and 6, thereby preventing their interaction with D cyclins. We have recently provided evidence that the gene is a tumor suppressor for myeloid leukemia in mice. First, retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation within the gene's promoter region. Application of the bisulfite sequencing protocol and methylation specific PCR demonstrated that 53 percent of primary leukmias were positive for hypermethylation within the CpG island covering the 5' region containing regulatory sequences. In some cases where leukemias did not have hypermethylation in the primary state, the neoplastic cells developed methylation following transplantation in syngeneic mice. The epigenetic change correlated with decreased expression in primary and transplanted leukemias as shown by TaqMan real-time RT-PCR. Furthermore, treatment of a leukemia cell line that had Ink4b hypermethylation, with the methylation inhibitor 5-aza-2' deoxycytidine, resulted in an increase in p15Ink4b mRNA expression. A second type of study carried out in p15Ink4b-deficient mice provided the most convincing data regarding the tumor suppressor role of Ink4b. Use of the mice in a retrovirus-induced leukemogenesis protocol showed that mice heterozygous for Ink4b deficiency had increased susceptibility to myeloid leukemia. Mice were inoculated with retrovirus MOL4070LTR, a unique myeloid disease-inducing virus, which was recently constructed and produced in our laboratory. The retrovirus provided the cooperating events through insertional mutagenesis that allowed the demonstration of the susceptibility to leukemia provided by loss of one allele. Mice deficient in both alleles were not more susceptible than those deficient in one allele, raising the possibility that there are opposing forces to the development of myeloid leukemia in Ink4b null mice. Our laboratory has shown that Ink4b expression at the mRNA level is up-regulated following treatment with cytokines such as interferon (IFN), TGFbeta, and IL-6 that are found in hematopoietic and immune environments. Because this is an important way that cells are instructed to growth arrest, we have also become interested in determining the elements in the promoter that control Ink4b expression in the context of cytokine induction. As a part of an investigation into the mechanisms involved in IFNbeta-induced expression of p15Ink4b mRNA in the murine myeloid cell line M1, we determined that both the interferon consensus sequence binding protein (ICSBP)/IRF-8 and the Ets-like protein PU.1 were able to activate transcription from Ink4b promoter sequences linked to a luciferase reporter gene. A 330 bp fragment located 116 bp upstream of the translation initiation site was sufficient for this activity and there was evidence that the effect of these factors was additive. Analysis of ICSBP mutants indicated that its DNA binding region and its PU.1 interaction domain are necessary to stimulate transcription and specific ICSBP and PU.1binding sites (-350 and -320, respectively) were identified. Our data suggest that binding of both factors to DNA is required in order to stabilize the PU.1/ICSBP binding complex. This regulation of the Ink4b promoter is apparently myeloid-specific, because both ICSBP and PU.1 are myeloid commitment factors. Interestingly, our study on transcriptional regulation of Ink4b provides, at least in part, an explanation for the tumor suppressor activity of ICSBP demonstrated in ICSBP-deficient mice. These mice develop a chronic myelogenous leukemia (CML)-like disease and this is consistant with the finding that a high percentage of human AMLs and CMLs lack ICSBP transcripts. Our ongoing studies are aimed at determining genes that collaborate with inactivation of Ink4b, determining the effects of p15Ink4b on normal hematopoiesis, and investigating further regulation of Ink4b transcription through transcription factors and methylation.